4′-C-substituted-2-haloadenosine derivative

ABSTRACT

The present invention provides a 4′-C-substituted-2-haloadenosine derivative represented by the following formula [I], [II], or [III]: 
                         
(wherein X represents a halogen atom, R 1  represents an ethynyl group or a cyano group, and R 2  represents hydrogen, a phosphate residue, or a phosphate derivative residue). The present invention also provides a pharmaceutical composition containing the derivative and a pharmaceutically acceptable carrier therefor. Such derivative is useful as medicine for the treatment of Acquired Immune Deficiency Syndrome (AIDS).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to 4′-C-substituted-2-haloadenosinederivatives and use thereof as a medicine, in particular a medicinewhich is useful for the treatment of acquired immunodeficiency syndrome(AIDS).

2. Background Art

The clinical setting for AIDS has been dramatically changed by amulti-drug combination therapy, which is sometimes called highly activeantiretroviral therapy, or HAART. In HAART, nucleoside reversetranscriptase inhibitors (NRTIs) such as zidovudine (AZT), didanosine(ddI), zalcitabine (ddC), stavudine (d4T), and lamivudine (3TC), andprotease inhibitors (PIs) are employed in combination.

Although HAART has drastically decreased the number of deaths caused byAIDS, there has emerged a multi-drug resistant HIV-1 (humanimmunodeficiency virus-1) mutant exhibiting cross-resistance to variousdrugs. For example, in the early 1990s patients infected with an HIVexhibiting resistance to both AZT and 3TC were very rare, whereas in1995-1996 the percentage of AIDS patients infected with such an HIVbecame as high as 42%.

Ohrui, et al. have synthesized 2′-deoxy-4′-C-ethynyl nucleosides andassayed the anti-HIV activity thereof, and as a result, have found thata 2′-deoxy-4′-C-ethynyl nucleoside having a specific structure exhibitspotent anti-HIV activity equal to or higher than that of AZT, and haseffective antiviral activity against a multi-drug-resistant viral strainexhibiting resistance to various anti-HIV drugs such as AZT, ddI, ddC,d4T, and 3TC. (See, for example, Nucleic Acids Symp. Ser., January 2000,(44): 105-6; J. Med. Chem., November 2000, 43(23): 4516-25; Curr. DrugTargets Infect. Disord, May 2001, 1(1): 1-10; Antimicrob. AgentsChemother., May 2001, 45: 1539-1546; Nucleosides Nucleotides NucleicAcids, May 2003, 22(5-8): 887-9; WO 00/69876; WO 00/69877; and WO03/68796.)

The present inventors have evaluated in vitro toxicity of 4′-C-ethynylpurine nucleoside derivatives and 4′-C-cyano purine nucleosidederivatives, which, among a variety of 4′-C-substituted nucleosides,exhibit particularly potent anti-HIV activity. As a result, the presentinventors have found that: (1) 2,6-diaminopurine derivatives and guaninederivatives, which exhibit the most potent anti-HIV activity, exhibittoxicity in vitro and in vivo; and (2) adenine derivatives, whichexhibit less toxicity, are readily converted into hypoxanthinederivatives in blood by adenosine deaminase, thereby weakening theanti-HIV activity of the derivatives.

In order to attain further enhancement of selectivity index; i.e.,(concentration at which cytotoxicity is obtained)/(concentration atwhich anti-HIV activity is obtained) and to provide resistance toinactivation by adenosine deaminase, the present inventors havesynthesized a variety of derivatives through chemical modification of4′-C-substituted-2′-deoxyadenosine (a lead compound), which, amongvarious 4′-C-substituted purine nucleosides, exhibits potent anti-HIVactivity and less toxicity.

As has been known, when a halogen atom, which exhibits electronattraction, is introduced to the 2-position of the base moiety of anadenosine derivative, the resultant derivative exhibits a certain levelof resistance to inactivation by adenosine deaminase (Chem. Pharm.Bull., 42(1994), p 1688; J. Med. Chem., 39(1996), p 3847). However,whether or not selectivity index can be improved through introduction ofa halogen atom has remained unknown.

Only one literature discloses that introduction of an ethynyl group tothe 4′-position of d4T (stavudine: 2′,3′-didehydro-3′-deoxythymidine)enhances the selectivity index of d4T (Bioorg. Med. Chem. Lett.,November 2003, 13(21): 3775-7). However, effects similar to those of d4Tare not expected to be obtained in an adenosine derivative, which is apurine nucleoside, whose basic skeleton differs considerably from thatof d4T, and therefore, this literature does not provide usefulinformation for the present inventors' purposes.

SUMMARY OF THE INVENTION

The present inventors have performed studies on the anti-HIV activity,etc. of the newly synthesized derivatives, and have found that2′-deoxy-4′-C-ethynyl-2-fluoroadenosine—which is obtained by introducinga fluorine atom to the 2-position of the base moiety of2′-deoxy-4′-C-ethynyladenosine (i.e., lead compound)—exhibits resistanceto inactivation by adenosine deaminase, has potent antiviral activityagainst a multi-drug-resistant virus strain exhibiting resistance tovarious anti-HIV drugs such as AZT, ddI, ddC, d4T, and 3TC, and exhibitsenhanced anti-HIV activity and considerably lowered cytotoxicity.

On the basis of this finding, the present inventors have synthesized avariety of 4′-C-substituted-2-haloadenosine derivatives, each beingformed of 2-haloadenine (base moiety) and a sugar moiety having anethynyl or cyano group at the 4-position, and have assayed biologicalactivities of the thus-synthesized derivatives. The present inventionhas been accomplished on the basis of the results of the assay.

Accordingly, the present invention provides a4′-C-substituted-2-haloadenosine derivative represented by the followingformula [I], [II], or [III]:

(wherein X represents a halogen atom, R¹ represents an ethynyl group ora cyano group, and R² represents hydrogen, a phosphate residue, or aphosphate derivative residue).

The present invention also provides a pharmaceutical compositioncontaining the 4′-C-substituted-2-haloadenosine derivative and apharmaceutically acceptable carrier therefor.

The present invention also provides a method of treating AIDS,comprising administering, to a human or an animal, the4′-C-substituted-2-haloadenosine derivative or a pharmaceuticalcomposition containing the derivative.

As shown in the Test Examples provided hereinbelow, the compounds of thepresent invention (e.g., 2′-deoxy-4′-C-ethynyl-2-fluoroadenosine)exhibit resistance to inactivation by adenosine deaminase, have potentantiviral activity against a multi-drug-resistant virus strainexhibiting resistance to various anti-HIV drugs such as AZT, ddI, ddC,d4T, and 3TC, exhibit unexpectedly enhanced anti-HIV activity;specifically, anti-HIV activity higher by a factor of 144 than that of2′-deoxy-4′-C-ethynyladenosine (i.e., lead compound), and exhibitconsiderably lowered cytotoxicity. Therefore, surprisingly, thecompounds of the present invention exhibit a selectivity index of110,000, which is considerably higher than that of2′-deoxy-4′-C-ethynyladenosine (EdAdo) (i.e., 1,630).

As described above, the compounds of the present invention exhibitexcellent anti-HIV activity, particularly against a multi-drug-resistantHIV strain having resistance to various anti-HIV drugs such as AZT, DDI,DDC, D4T, and 3TC, exhibit less cytotoxicity, and exhibit resistance toinactivation by adenosine deaminase. Therefore, the compounds of thepresent invention are envisaged for development for producingpharmaceuticals, particularly drugs for treating AIDS.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows stability of compounds against deamination reaction inducedby adenosine deaminase. The black squares show the results obtained from2′-deoxy-4′-C-ethynyl-2-fluoroadenosine (a compound of the presentinvention), whereas the black circles show the results obtained from2′-deoxy-4′-C-ethynyladenosine (a known compound);

FIG. 2 shows stability of 2′-deoxy-4′-C-ethynyl-2-fluoroadenosine (acompound of the present invention) under acidic conditions;

FIG. 3 shows stability of 2′,3′-dideoxyadenosine (ddAdo; a knowncompound) under acidic conditions; and

FIG. 4 shows changes in body weight of mice, as measured afteradministration of 2′-deoxy-4′-C-ethynyl-2-fluoroadenosine (a compound ofthe present invention). In FIG. 4, Graph A shows the results obtainedfrom oral administration, and graph B shows the results obtained fromintravenous injection. In both graphs, white circles show the resultsfrom placebo administration, and triangles and squares correspond to adose of 30 mg/kg and 100 mg/kg, respectively.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(1) Compounds

The compounds of the present invention are represented by formulas [I],[II], and [III]. Examples of the phosphate residue represented by R² inthese formulas include a monophosphate residue, a diphosphate residue, atriphosphate residue, and a phosphonate; and examples of the phosphatederivative residue include phosphate polyesters (e.g., a phosphatediester and a phosphate triester), phosphate amidates (e.g., a phosphatemonoamidate and a phosphate diamidate), phosphorothioate,phosphoroselenoate, and phosphoroboranoate. Examples of halogen atomsrepresented by X include bromine, iodine, fluorine, and chlorine.

Of these compounds, preferred ones are those that satisfy one or more ofthe following requirements: (a) R² is hydrogen or phosphonate; (b) X isfluorine or chlorine; and (c) R¹ is an ethynyl group. Specific examplesof preferred compounds are given below:

<Compounds Represented by Formula [I]>

2′-deoxy-4′-C-ethynyl-2-fluoroadenosine,4′-C-cyano-2′-deoxy-2-fluoroadenosine,2-chloro-2′-deoxy-4′-C-ethynyladenosine, and2′-deoxy-4′-C-ethynyl-2-fluoroadenosine 5′-H-phosphonate;

<Compounds Represented by Formula [II]>

2′,3′-didehydro-2′,3′-dideoxy-4′-C-ethynyl-2-fluoroadenosine,2′,3′-didehydro-2′,3′-dideoxy-4′-C-cyano-2-fluoroadenosine,2′,3′-didehydro-2′,3′-dideoxy-4′-C-ethynyl-2-chloroadenosine, and2′,3′-didehydro-2′,3′-dideoxy-4′-C-ethynyl-2-fluoroadenosine5′-H-phosphonate; and

<Compounds Represented by Formula [III]>

2′,3′-dideoxy-4′-C-ethynyl-2-fluoroadenosine,2′,3′-dideoxy-4′-C-cyano-2-fluoroadenosine,2′,3′-dideoxy-4′-C-ethynyl-2-chloroadenosine, and2′,3′-dideoxy-4′-C-ethynyl-2-fluoroadenosine 5′-H-phosphonate.

The compounds of the present invention may be salts, hydrates, orsolvates. When R² is hydrogen, examples of salts include acid-adductssuch as hydrochlorides and sulfates; and when R² is a phosphate residue,examples of salts include alkali metal salts such as sodium salts,potassium salts, and lithium salts; alkaline earth metal salts such ascalcium salts; and ammonium salts, and any of those salts may be used solong as they are pharmaceutically acceptable.

Examples of hydrates or solvates include adducts formed by combining onemolecule of the compound of the present invention or a salt thereof and0.1-3.0 molecules of water or a solvent. In addition, the compounds ofthe present invention encompass a variety of isomers thereof such astautomers.

(2) Production Method

The compounds [I] of the present invention can be produced through thebelow-described steps.

First Step:

In the first step, hydroxyl groups at the 3′- and 5′-positions of acompound represented by formula [IV] are protected, to thereby yield acompound represented by formula [V]:

(wherein P represents a protective group, and R¹ represents an ethynylgroup or a cyano group).

The compound [IV] (i.e., starting material) is a known compound;specifically, a compound in which R¹ is an ethynyl group (J. Med. Chem.,43, 4516-4525 (2000)), or a compound in which R¹ is a cyano group (WO03/68796).

The protective groups represented by P, which protect the hydroxylgroups at the 3′- and 5′-positions, may be those groups which aregenerally employed for protecting a hydroxyl group. Examples of types ofthe protective groups include an ether type, an acyl type, a silyl type,and an acetal type. Specific examples of the protective groups which maybe employed include ether-type protective groups such as methyl ether,tert-butyl ether, benzyl ether, methoxybenzyl ether, and trityl ether;acyl-type protective groups such as acetyl, benzoyl, and pivaloyl;silyl-type protective groups such as t-butyldimethylsilyl,t-butyldiphenylsilyl, trimethylsilyl, and triethylsilyl; and acetal-typeprotective groups such as isopropylidene, ethylidene, methylidene,benzylidene, tetrahydropyranyl, and methoxymethyl.

Introduction of a protecting group is performed by conventional methods.For examples, in organic solvent such as pyridine, acetonitrile ordimethylformamide, compound [IV] is allowed to react with a protectingagent (alkyl halide, acid halide, acid anhydride or alkylsilyl halide)in the presence of a base such as metal alkoxide, triethylamine,4-dimethylaminopyridine or imidazole, at −10 to 100° C.

Second Step:

In the second step, the amino group at the 2-position of the compound[V] is converted into a halogen atom, to thereby yield a compoundrepresented by formula [VI]:

(wherein P represents a protective group, X represents a halogen atom,and R¹ represents an ethynyl group or a cyano group).

The compound [VI] can be synthesized through the following procedure:after the amino group at the 2-position of the compound [V] is treatedwith a nitrite derivative, halogen atom is introduced at the 2-positionof the base moiety by use of a halogen reagent; or the amino groups atthe 2- and 6-positions are treated under the same conditions, therebyforming a 2,6-dihalopurine derivative, and the halogen atom at the6-position of the base moiety is converted into an amino group throughtreatment with ammonia.

Examples of reagents for substituting the amino group at the 2-positionof the compound [V] by fluorine include sodium nitrite intetrafluoroboric acid; and a nitrous acid ester (e.g., t-butyl nitrite)in hydrogen fluoride-pyridine.

Reaction conditions vary depending on the reagent employed. For example,when t-butyl nitrite is employed in hydrogen fluoride-pyridine, t-butylnitrite (1 to 3 mol) is added to the compound [V] in hydrogenfluoride-pyridine serving as a solvent, and the resultant mixture isallowed to react at −50° C. to room temperature for about 15 minutes toabout five hours. When the compound [V] is formed into a2,6-difluoropurine derivative, the resultant derivative is treated withaqueous ammonia in an organic solvent such as dioxane or methanol.

Examples of reagents for substituting the amino group at the 2-positionof the compound [V] by chlorine include a combination of antimonytrichloride and t-butyl nitrite, and a combination of acetyl chlorideand benzyltriethylammonium nitrite, which combinations are employed inan organic solvent such as dichloromethane.

Reaction conditions vary depending on the reagent employed. For example,when a combination of acetyl chloride and benzyltriethylammonium nitriteis employed as the reagent, in an organic solvent such asdichloromethane, benzyltriethylammonium nitrite (1 to 5 mol) is treatedwith acetyl chloride (1 to 5 mol) at −50° C. to room temperature forabout 30 minutes to about three hours, and the resultant mixture isallowed to react with the compound [V] (1 mol) at −50° C. to roomtemperature for one hour to a few days. When the compound [V] is formedinto a 2,6-dichloropurine derivative, the resultant derivative istreated with aqueous ammonia in an organic solvent such as dioxane ormethanol.

The protective groups of the thus-obtained compound [VI] are removed, tothereby yield the compound of the present invention in which R² ishydrogen, and if desired, the compound is phosphorylated:

(wherein P represents a protective group, X represents a halogen atom,R¹ represents an ethynyl group or a cyano group, and R² representshydrogen, a phosphate residue, or a phosphate derivative residue).

The protective groups may be removed through a technique which isappropriately selected from among typical techniques (e.g., hydrolysisunder acidic conditions, hydrolysis under alkaline conditions, treatmentwith tetrabutylammonium fluoride, and catalytic reduction) in accordancewith the protective groups employed.

(wherein X represents a halogen atom, R¹ represents an ethynyl group ora cyano group, and R² represents hydrogen).

In order to produce the 5′-H-phosphonate derivative [VII] (the compoundof the present invention), the compound [I] in which R² is hydrogen andphosphonic acid are subjected to condensation in an organic solvent byuse of an appropriate condensing agent. Examples of the organic solventwhich may be employed include pyridine, and dimethylformamide in thepresence of a base such as triethylamine. Examples of the condensingagent which may be employed include carbodiimides such as dicyclohexylcarbodiimide, diisopropyl carbodiimide, and water-soluble carbodiimide;sulfonic acid halides such as toluenesulfonyl chloride; and phosphatechlorides such as diphenyl phosphate chloride.

Reaction conditions vary depending on the reagent employed. For example,when dicyclohexyl carbodiimide is employed in pyridine, phosphonic acid(1 to 5 mol) and dicyclohexyl carbodiimide (1 to 10 mol) are added to 1mol of the compound [1], and the resultant mixture is allowed to reactat 0° C. to 50° C. for about one to about 24 hours.

When a compound in which R² is a monophosphate is to be produced, acompound in which R² is hydrogen is reacted with a phosphorylatingagent; for example, phosphorus oxychloride or tetrachloropyrophosphoricacid, which selectively phosphorylates the 5′-position of a nucleoside.When a compound in which R² is a diphosphate or triphosphate is to beproduced, the corresponding 5′-monophosphate compound is activated inthe form of phosphoimidazolide, phosphomorpholidate, or anhydrousdiphenylphosphate, and the thus-activated compound is reacted withphosphoric acid, pyrophosphoric acid, or a suitable salt thereof, tothereby produce a target compound in a free acid or salt form.

The compounds [II] of the present invention can be produced through thebelow-described steps.

First Step:

In the first step, the hydroxyl group at the 5′-position of a compoundrepresented by formula [I] in which R² is hydrogen is selectivelyprotected, to thereby yield a compound represented by formula [VIII]:

(wherein P represents a protective group, X represents a halogen atom,R¹ represents an ethynyl group or a cyano group, and R² representshydrogen).

The protective group represented by P, which protects the hydroxyl groupat the 5′-position, may be a protective group which is generallyemployed for selectively protecting a primary hydroxyl group. Specificexamples of the protective group include a trimethoxytrityl group, adimethoxytrityl group, a methoxytrityl group, a trityl group, at-butyldimethylsilyl group, a t-butyldiphenylsilyl group, and a benzoylgroup.

Introduction of the protective group can be carried out in a mannersimilar to that employed for the compound [V].

Second Step:

In the second step, the hydroxyl group at the 3′-position of thecompound [VIII] is subjected to dehydration, forming a2′,3′-carbon-carbon double bond, to thereby yield a compound representedby formula [VIV].

(wherein P represents a protective group, X represents a halogen atom,and R¹ represents an ethynyl group or a cyano group).

In order to produce the compound [VIV] through dehydration of thehydroxyl group at the 3′-position of the compound [VIII], the hydroxylgroup at the 3′-position of the compound [VIII] is converted into aremovable functional group such as a sulfonate group (e.g., amethanesulfonate group, a chloromethanesulfonate group, atoluenesulfonate group, or a trifluoromethanesulfonate group) or ahalogen atom, and the thus-converted group is removed through treatmentwith a base.

Reaction conditions vary depending on the reagent employed. For example,in the case of reaction through formation of atrifluoromethanesulfonate, trifluoromethanesulfonic anhydride (1 to 5mol) and a base (e.g., pyridine or triethylamine) (5 to 10 mol) areadded to the compound [VIII] in an organic solvent such asdichloromethane or pyridine, and the resultant mixture is allowed toreact at −78° C. to room temperature for about one to about 24 hours.

The protective group of the thus-obtained compound [VIV] is removed, tothereby yield the compound of the present invention in which R² ishydrogen, and if desired, the compound is phosphorylated:

(wherein X represents a halogen atom, P represents a protective group,R¹ represents an ethynyl group or a cyano group, and R² representshydrogen, a phosphate residue, or a phosphate derivative residue).

The protective group may be removed through a technique which isappropriately selected from among typical techniques (e.g., hydrolysisunder acidic conditions, hydrolysis under alkaline conditions, treatmentwith tetrabutylammonium fluoride, and catalytic reduction) in accordancewith the protective group employed.

A compound in which R² is a phosphate residue or a derivative thereofcan be synthesized in a manner similar to that of the compound [I].

The compounds [III] of the present invention can be produced through thebelow-described steps.

First Step:

In the first step, the hydroxymethyl group at the 4-position of acompound represented by formula [X] is oxidized to thereby form analdehyde group, which is further converted into a triethylsilylethynylor cyano group to thereby yield a compound represented by formula [XI]:

(wherein R¹ represents an ethynyl group, a triethylsilylethynyl group,or a cyano group).

The compound [X] (i.e., starting material) is a known compound (Biosci.Biotech. Biochem., 57, 1433-1438 (1993)). The compound [X] can beconverted into a triethylsilylethynyl compound through the followingprocedure: the hydroxymethyl group at the 4-position of the compound [X]is oxidized to form a formyl group, and the formyl group is convertedinto a dibromovinyl group, followed by removal of hydrogen bromidethrough treatment with a strong base.

When the hydroxymethyl group at the 4-position of the compound [X] isconverted into a formyl group, an oxidizing agent is employed. Examplesof the oxidizing agent which may be employed include chromium-containingoxidizing agents such as chromic anhydride-pyridine-acetic anhydridecomposite reagents, pyridinium chlorochromate, and pyridiniumdichromate; high-valency iodine oxidizing agents such as Dess-Martinreagent; and dimethyl sulfoxide-based oxidizing agents such as acombination of dimethyl sulfoxide and acetic anhydride, oxalyl chloride,or dicyclohexyl carbodiimide.

Reaction conditions vary depending on the oxidizing agent to beemployed. For example, when oxidation is carried out by use of oxalylchloride and dimethyl sulfoxide, oxalyl chloride (1 to 5 mol) anddimethyl sulfoxide (1.5 to 6 mol) are added to 1 mol of the compound [X]in an organic solvent (e.g., dichloromethane), optionally under an inertgas atmosphere (e.g., argon or nitrogen), and the resultant mixture isallowed to react at −100° C. to 0° C. for about 15 minutes to about twohours. Subsequently, a base such as triethylamine is added in an amountof 2 to 10 mol to the mixture, and the resultant mixture is furtherallowed to react at room temperature for about 15 minutes to about twohours.

The thus-formed aldehyde can be converted into a corresponding alkynethrough the following procedure: the aldehyde is subjected tocarbon-increasing (i.e., C—C bond formation) reaction; the resultantcompound is treated with a strong base to thereby form a metal alkynylcompound; and a protective group is introduced into the metal alkynylcompound. Carbon-increasing reaction is carried out in an organicsolvent such as dichloromethane or dichloroethane, optionally under aninert gas atmosphere (e.g., argon or nitrogen). Specifically, carbontetrabromide (1 to 5 mol) and triphenylphosphine (2 to 10 mol) are addedto 1 mol of the above-formed aldehyde, and the resultant mixture isallowed to react at 0 to 50° C. for about 15 minutes to about threehours.

Treatment with a strong base can be carried out in an organic solventsuch as tetrahydrofuran, 1,4-dioxane, or dimethoxyethane, optionallyunder an inert gas atmosphere (e.g., argon or nitrogen). Specifically, alithium compound (e.g., methyllithium, n-butyllithium, ort-butyllithium) (2 to 4 mol) is added to 1 mol of the compound obtainedthrough carbon-increasing reaction, and the resultant mixture is allowedto react at −100 to −20° C. for about five to about 60 minutes.Furthermore, when a silyl protective group is introduced into thealkynyl group of the thus-obtained compound, the aforementionedstrong-base treatment is followed by addition of a silylating agent suchas chlorotriethylsilane, and the resultant mixture is allowed to react.

Meanwhile, the compound [X] can be converted into a cyano compoundthrough the following procedure: the hydroxymethyl group at the4-position of the compound [X] is oxidized to form a formyl group, andthe formyl group is converted into an oxime group, followed bydehydration of the thus-formed oxime group.

When the hydroxymethyl group at the 4-position of the compound [X] isconverted into a formyl group, an oxidizing agent is employed. Examplesof the oxidizing agent which may be employed include chromium-containingoxidizing agents such as chromic anhydride-pyridine-acetic anhydridecomposite reagents, pyridinium chlorochromate, and pyridiniumdichromate; high-valency iodine oxidizing agents such as Dess-Martinreagent; and dimethyl sulfoxide-based oxidizing agents such as acombination of dimethyl sulfoxide and acetic anhydride, oxalyl chloride,or dicyclohexyl carbodiimide.

Reaction conditions vary depending on the oxidizing agent to beemployed. For example, when oxidation is carried out by use of oxalylchloride and dimethyl sulfoxide, oxalyl chloride (1 to 5 mol) anddimethyl sulfoxide (1.5 to 6 mol) are added to 1 mol of the compound [X]in an organic solvent (e.g., dichloromethane), optionally under an inertgas atmosphere (e.g., argon or nitrogen), and the resultant mixture isallowed to react at −100° C. to 0° C. for about 15 minutes to about twohours. Subsequently, a base such as triethylamine is added in an amountof 2 to 10 mol to the mixture, and the resultant mixture is furtherallowed to react at room temperature for about 15 minutes to about twohours.

The thus-formed aldehyde can be converted into a corresponding oxime byreacting 1 mol of the aldehyde with hydroxylamine hydrochloride (1 to 5mol) in an organic solvent such as pyridine at room temperature to 100°C. for about 30 minutes to about three hours.

Dehydration of the thus-formed oxime can be carried out by use of adehydrating agent (e.g., phosgene, carbonyldiimidazole, methanesulfonylchloride, or acetic anhydride) in an organic solvent (e.g.,dichloromethane, acetonitrile, or tetrahydrofuran) in the presence of abase (e.g., pyridine, triethylamine, or sodium acetate).

Dehydration conditions vary depending on the dehydrating agent to beemployed. For example, when dehydration is carried out by use ofmethanesulfonyl chloride, in an organic solvent (such asdichloromethane, tetrahydrofuran, or pyridine), methanesulfonyl chloride(1 to 5 mol) and triethylamine (5 to 10 mol) are added to 1 mol of theoxime, and the resultant mixture is allowed to react at −50° C. to roomtemperature for about 15 minutes to about two hours.

Second Step:

In the second step, the methoxybenzylidene group which protects thehydroxyl groups at the 3- and 5-positions of the compound [XI] isremoved, to thereby yield a compound represented by formula [XII]:

(wherein R¹ represents an ethynyl group, a triethylsilylethynyl group,or a cyano group).

The protective group may be removed through a technique which isappropriately selected from among typical techniques (e.g., hydrolysisunder acidic conditions, and catalytic reduction).

Reaction conditions vary depending on the technique to be employed. Forexample, when the protective group is removed through hydrolysis underacidic conditions, the compound [XI] is allowed to react in an aqueoussolution of an organic acid (e.g., formic acid or acetic acid) ormineral acid at 0 to 100° C. for one to 24 hours.

Third Step:

In the third step, the hydroxyl group at the 5-position of the compound[XII] is selectively protected, to thereby yield a compound representedby formula [XIII]:

(wherein P represents a protective group, and R¹ represents an ethynylgroup, a triethylsilylethynyl group, or a cyano group).

The protective group represented by P, which protects the hydroxyl groupat the 5-position, may be a protective group which is generally employedfor selectively protecting a primary hydroxyl group. Specific examplesof the protective group include a trimethoxytrityl group, adimethoxytrityl group, a methoxytrityl group, a trityl group, at-butyldimethylsilyl group, a t-butyldiphenylsilyl group, and a benzoylgroup.

Introduction of the protective group can be carried out in a mannersimilar to that employed for the compound [V].

Fourth Step:

In the fourth step, the hydroxyl group at the 3-position of the compound[XIII] is reduced, to thereby yield a compound represented by formula[XIV]:

(wherein P represents a protective group, and R¹ represents an ethynylgroup, a triethylsilylethynyl group, or a cyano group).

Deoxygenation of the hydroxyl group at the 3-position can be carried outby converting the compound having the hydroxyl group into acorresponding halide (iodite, bromide or chloride),phenoxythionocarbonate, thiocarbonylimidazole, or methyldithiocarbonate,and by reducing the thus-converted compound by use of a radical reducingagent in the presence of a radical initiator.

For example, when deoxygenation is carried out through formation of aphenoxythiocarbonyl compound, conversion of the hydroxyl group to aphenoxythiocarbonyl group is carried out in an organic solvent (e.g.,tetrahydrofuran, acetonitrile, or dichloromethane) in the presence of abase such as dimethylaminopyridine or pyridine, optionally under aninert gas atmosphere such as argon or nitrogen. Specifically, a phenylchlorothionoformate derivative (1 to 10 mol, preferably 1 to 2 mol) isadded to 1 mol of the aforementioned compound in which only theprotective group for the hydroxyl group at the 3-position has beeneliminated, and the resultant mixture is allowed to react under stirringat 0 to 50° C. for about 0.5 to about five hours.

Subsequently, reduction is carried out in an organic solvent (e.g.,toluene or benzene) in the presence of a radical initiator such asazobisisobutyronitrile, optionally under an inert gas atmosphere such asargon or nitrogen. Specifically, a radical reducing agent such astributyltin hydride or tris(trimethylsilyl)silane (1 to 10 mol,preferably 2 to 5 mol) is added to 1 mol of the aforementionedphenoxythiocarbonyl compound, and the resultant mixture is allowed toreact under stirring at 50 to 150° C. for about one to about five hours.

Fifth Step:

In the fifth step, the isopropylidene group at the 1- and 2-positions ofthe compound [XIV] is removed, and then the thus-formed hydroxyl groupsare acetylated, to thereby yield a compound represented by formula [XV]:

(wherein P represents a protective group, and R¹ represents an ethynylgroup, a triethylsilylethynyl group, or a cyano group).

When the isopropylidene group at the 1- and 2-positions is removedthrough hydrolysis under acidic conditions, the compound [XIV] isallowed to react in an aqueous solution of an organic acid (e.g., formicacid or acetic acid) or mineral acid at 0 to 100° C. for one to 24hours.

Introduction of acetyl groups to the hydroxyl groups, which followsremoval of the isopropylidene group, can be carried out by means of acustomary technique. For example, acetyl groups are introduced to thehydroxyl groups through reaction with an acetylating agent (e.g., acetylchloride or acetic anhydride) in an organic solvent such as pyridine,acetonitrile, or dichloromethane in the presence of a base such aspyridine or triethylamine.

For example, in the case of reaction in pyridine by use of aceticanhydride, acetic anhydride (2 to 10 mol) and, if desired, a catalyticamount of 4-dimethylaminopyridine are added to 1 mol of the compoundfrom which the isopropylidene group has been removed, and the resultantmixture is allowed to react at 0 to 100° C. for one to 24 hours.

Sixth Step:

In the sixth step, the compound [XV] and 2,6-diaminopurine are subjectedto condensation, to thereby yield a compound represented by formula[XVI]:

(wherein P represents a protective group, and R¹ represents an ethynylgroup, a triethylsilylethynyl group, or a cyano group).

Condensation of the compound [XV] and 2,6-diaminopurine can be carriedout by reacting the compound [XV] with 2,6-diaminopurine in the presenceof a Lewis acid. In this case, 2,6-diaminopurine may be silylated, andsuch silylation of 2,6-diaminopurine may be carried out through a knowntechnique. For example, 2,6-diaminopurine is silylated under reflux in amixture of hexamethyldisilazane and trimethylchlorosilane, or issilylated under reflux by use of bis(trimethylsilyl)acetamide in anorganic solvent such as acetonitrile or 1,2-dichloroethane. Examples ofLewis acids to be employed include trimethylsilyltrifluoromethanesulfonate, tin tetrachloride, zinc chloride, zinciodide, and anhydrous aluminum chloride.

Condensation reaction can be carried out in an organic solvent such asdichloromethane, 1,2-dichloroethane, acetonitrile, or toluene,optionally under an inert gas atmosphere such as argon or nitrogen.Specifically, 2,6-diaminopurine (1 to 10 mol) and a Lewis acid (0.1 to10 mol) are added to 1 mol of the compound [XV], and the resultantmixture is allowed to react at −20 to 150° C. for about 30 minutes toabout 24 hours.

Seventh Step:

In the seventh step, the amino group at the 2-position of the compound[XVI] is converted into halogen atom, to thereby yield a compoundrepresented by formula [XVII]:

(wherein P represents a protective group, X represents a halogen atom,and R¹ represents an ethynyl group, a triethylsilylethynyl group, or acyano group).

The compound [XVII] can be synthesized through the following procedure:after the amino group at the 2-position of the compound [XVI] is treatedwith a nitrite derivative, halogen atom is introduced at the 2-positionof a base moiety by use of a halogen reagent; or the amino groups at the2- and 6-positions are treated under the same conditions, therebyforming a 2,6-dihalopurine derivative, and the halogen atom at the6-position of base moiety is converted into an amino group throughtreatment with ammonia.

Examples of reagents for substituting the amino group at the 2-positionof the compound [XVI] by fluorine include sodium nitrite intetrafluoroboric acid; and a nitrous acid ester (e.g., t-butyl nitrite)in hydrogen fluoride-pyridine.

Reaction conditions vary depending on the reagent employed. For example,when t-butyl nitrite is employed in hydrogen fluoride-pyridine, t-butylnitrite (1 to 3 mol) is added to the compound [XVI] in hydrogenfluoride-pyridine serving as a solvent, and the resultant mixture isallowed to react at −50° C. to 0° C. for about 15 minutes to about fivehours. When the compound [XVI] is formed into a 2,6-difluoropurinederivative, the resultant derivative is treated with aqueous ammonia inan organic solvent such as dioxane or methanol.

Examples of reagents for substituting the amino group at the 2-positionof the compound [XVI] by chlorine include a combination of antimonytrichloride and t-butyl nitrite, and a combination of acetyl chlorideand benzyltriethylammonium nitrite, which combinations are employed inan organic solvent such as dichloromethane.

Reaction conditions vary depending on the reagent employed. For example,when a combination of acetyl chloride and benzyltriethylammonium nitriteis employed as the reagent, in an organic solvent such asdichloromethane, benzyltriethylammonium nitrite (1 to 5 mol) is treatedwith acetyl chloride (1 to 5 mol) at −50° C. to room temperature forabout 30 minutes to about three hours, and the resultant mixture isallowed to react with 1 mol of the compound [XVI] at −50° C. to roomtemperature for one hour to a few days. When the compound [XVI] isformed into a 2,6-dichloropurine derivative, the resultant derivative istreated with aqueous ammonia in an organic solvent such as dioxane ormethanol.

Eighth Step:

In the eighth step, the acetyl group which protects the hydroxyl groupat the 2′-position of the compound [XVII] is removed, to thereby yield acompound represented by formula [XVIII]:

(wherein P represents a protective group, X represents a halogen atom,and R¹ represents an ethynyl group, a triethylsilylethynyl group, or acyano group).

The acetyl group can be removed by use of an appropriate base or acidcatalyst. For example, when removal of the acetyl group is carried outin a solvent mixture of water and an alcohol (e.g., ethanol), a basecatalyst such as sodium hydroxide, potassium hydroxide, triethylamine,or aqueous ammonia can be employed.

For example, the acetyl group can be removed by allowing the compound[XVII] to react by use of aqueous ammonia in methanol at 0 to 100° C.for one to 24 hours.

Ninth Step:

In the ninth step, the hydroxyl group at the 2′-position of the compound[XVIII] is reduced, to thereby yield a compound represented by formula[XIX]:

(wherein P represents a protective group, X represents a halogen atom,and R¹ represents an ethynyl group, a triethylsilylethynyl group, or acyano group).

Deoxygenation of the hydroxyl group at the 3-position can be carried outby converting the compound having the hydroxyl group into thecorresponding halide (iodite, bromide or chloride),phenoxythionocarbonate, thiocarbonylimidazole, or methyldithiocarbonate,and by reducing the thus-converted compound by use of a radical reducingagent in the presence of a radical initiator.

For example, when deoxygenation is carried out through formation of aphenoxythiocarbonyl compound, conversion of the hydroxyl group to aphenoxythiocarbonyl group is carried out in an organic solvent (e.g.,tetrahydrofuran, acetonitrile, or dichloromethane) in the presence of abase such as dimethylaminopyridine or pyridine, optionally under aninert gas atmosphere such as argon or nitrogen. Specifically, a phenylchlorothionoformate derivative (1 to 10 mol, preferably 1 to 2 mol) isadded to 1 mol of the aforementioned compound in which only theprotective group for the hydroxyl group at the 2′-position has beeneliminated, and the resultant mixture is allowed to react under stirringat 0 to 50° C. for about 0.5 to about five hours.

Subsequently, reduction is carried out in an organic solvent (e.g.,toluene or benzene) in the presence of a radical initiator such asazobisisobutyronitrile, optionally under an inert gas atmosphere such asargon or nitrogen. Specifically, a radical reducing agent such astributyltin hydride or tris(trimethylsilyl)silane (1 to 10 mol,preferably 2 to 5 mol) is added to 1 mol of the aforementionedphenoxythiocarbonyl compound, and the resultant mixture is allowed toreact under stirring at 50 to 150° C. for about one to about five hours.

The protective group for the hydroxyl group of the thus-obtainedcompound [XIX] is removed, to thereby yield the compound of the presentinvention in which R² is hydrogen, and if desired, the compound isphosphorylated:

(wherein P represents a protective group, X represents a halogen atom,R¹ represents an ethynyl group, a triethylsilylethynyl group, or a cyanogroup, and R² represents hydrogen, a phosphate residue, or a phosphatederivative residue).

The protective group may be removed through a technique which isappropriately selected from among typical techniques (e.g., hydrolysisunder acidic conditions, hydrolysis under alkaline conditions, treatmentwith tetrabutylammonium fluoride, and catalytic reduction) in accordancewith the protective group employed.

A compound in which R² is a phosphate residue or a derivative thereofcan be synthesized in a manner similar to that of the compound [I].

The compounds of the present invention may be isolated and purifiedthrough conventional methods, in appropriate combination, which areemployed for isolating and purifying nucleosides and nucleotides; forexample, recrystallization, ion-exchange column chromatography, andadsorption column chromatography. The thus-obtained compounds mayfurther be converted to salts thereof in accordance with needs.

(3) Use

As shown in the below-described Test Examples, the compounds of thepresent invention exhibit excellent antiviral activity againstretroviruses. Thus, compositions of the present invention containing oneof the compounds of the present invention as an active ingredient findutility in the field of therapeutic drugs. Specifically, thecompositions of the present invention are useful for the treatment ofinfectious diseases caused by a retrovirus, in particular, AIDS, whichis caused by HIV infection.

The dose of the compounds of the present invention depends on and isdetermined in consideration of conditions such as the age, body weight,and type of disease of the patient; the severity of the disease; thedrug tolerance; and the administration route. However, the daily dose isdetermined to fall typically within 0.00001-1,000 mg/kg, preferably0.0001-100 mg/kg body weight. The compounds are administered in a singledose or divided doses.

Any administration route may be employed, and the compounds may beadministered orally, parenterally, enterally, or topically.

When a pharmaceutical is prepared from the compounds of the presentinvention, the compounds are typically mixed with customarily employedadditives, such as a carrier and an excipient. Examples of solidcarriers include lactose, kaolin, sucrose, crystalline cellulose, cornstarch, talc, agar, pectin, stearic acid, magnesium stearate, lecithin,and sodium chloride. Examples of liquid carriers include glycerin,peanut oil, polyvinylpyrrolidone, olive oil, ethanol, benzyl alcohol,propylene glycol, and water.

The product form is arbitrarily selected. When the carrier is solid,examples of product forms include tablets, powder, granules, capsules,suppositories, and troches, whereas when it is liquid, examples includesyrup, emulsion, soft-gelatin-capsules, cream, gel, paste, spray, andinjection.

EXAMPLES

The present invention will next be described in detail by way ofexamples including Synthesis Examples, Test Examples, and DrugPreparation Examples, which should not be construed as limiting theinvention thereto.

Synthesis Example 1 Synthesis of 2′-deoxy-4′-C-ethynyl-2-fluoroadenosine(Compound 4)

(1) Synthesis of9-(3,5-di-O-acetyl-2-deoxy-4-C-ethynyl-β-D-ribo-pentofuranosyl)-2,6-diaminopurine(Compound 2)

Compound 1 (0.33 g, 1.14 mmol) was suspended in acetonitrile (10.0 ml),and acetic anhydride (0.23 ml, 2.43 mmol), triethylamine (0.67 g, 4.81mmol), and a small amount of 4-dimethylaminopyridine were added to theresultant suspension, followed by stirring at room temperatureovernight.

The thus-precipitated crystals were filtered and dried, to thereby yieldcompound 2 (0.40 g, 1.07 mmol, 93.9%).

¹H-NMR(DMSO-d₆)δ7.94(1H, s, H-8), 6.76(2H, bs, NH₂), 6.27(1H, t, H-1′,J=7.00), 5.84(2H, bs, NH₂), 5.60(1H, dd, H-3′, J=4.00, 6.80), 4.46(1H,d, H-5′a, J=11.5), 4.21(1H, d, H-5′b, J=11.5), 3.74(1H, s, ethynyl)3.12(1H, m, H-2′a), 2.52(1H, m, H-2′b), 2.12, 2.03(each 3H, s, acetyl)

(2) Synthesis of 3′,5′-di-O-acetyl-2′-deoxy-4′-C-ethynyl-2-fluoroadenosine (Compound 3)

Compound 2 (450 mg, 1.20 mmol) was dissolved in 70% hydrogenfluoride-pyridine (5.00 ml), and t-butyl nitrite (0.194 ml, 1.63 mmol)was added to the resultant solution, followed by stirring at −10° C. forone hour. Distilled water was added to the resultant mixture, and theresultant mixture was subjected to extraction with chloroform. Theresultant organic layer was dried over anhydrous magnesium sulfate, andthen concentrated under reduced pressure. A mixture of chloroform andmethanol (50:1) was added to the resultant residue, and thethus-precipitated crystals were filtered and dried, to thereby yieldcompound 3 (240 mg, 0.64 mmol, 53.3%).

¹H-NMR(DMSO-d₆) δ8.34(1H, s, H-8), 7.94, 7.99(each 1H, bs, NH₂),6.35(1H, t, H-1′, J=6.80), 5.68(1H, dd, H-3′, J=5.10, 7.05), 4.41(1H, d,H-5′a, J=11.6), 4.21(1H, d, H-5′b, J=11.6), 3.42(1H, s, ethynyl),3.14(1H, m, H-2′a), 2.63(1H, m, H-2′b), 2.12, 2.00(each 3H, s, acetyl).

(3) Synthesis of 2′-deoxy-4′-C-ethynyl-2-fluoroadenosine (Compound 4)

Compound 3 (200 mg, 0.53 mmol) was dissolved in methanol (7.00 ml), and28% aqueous ammonia (5.00 ml) was added to the resultant solution,followed by stirring at room temperature for four hours. The resultantreaction mixture was concentrated under reduced pressure, and a mixtureof chloroform and methanol (20:1) was added to the resultant residue.The thus-precipitated crystals were filtered, and then the resultantcrystals were recrystallized from water, to thereby yield compound 4(113 mg, 0.39 mmol, 73.6%).

¹H-NMR(DMSO-d₆) δ8.30(1H, s, H-8), 7.87, 7.84(each 1H, bs, NH₂),6.24(1H, dd, H-1′, J=5.05, 7.15), 5.57(1H, d, 3′—OH, J=5.50), 5.30(1H,t, 5′—OH, J=6.40), 4.57(1H, m, H-3′), 3.65(1H, m, H-5′a), 3.55(1H, m,H-5′b), 3.51(1H, s, ethynyl), 2.70(1H, m, H-2′a), 2.44(1H, m, H-2′b).

Synthesis Example 2 Synthesis of 4′-C-cyano-2′-deoxy-2-fluoroadenosine(Compound 8)

(1) Synthesis of9-(3,5-di-O-acetyl-4-C-cyano-2-deoxy-β-D-ribo-pentofuranosyl)-2,6-diaminopurine(Compound 6)

Compound 5 (122 mg, 0.418 mmol) was suspended in acetonitrile (5.00 ml),and acetic anhydride (118 μl, 1.25 mmol), triethylamine (352 μl, 2.51mmol), and a small amount of 4-dimethylaminopyridine were added to theresultant suspension, followed by stirring at room temperatureovernight. The thus-precipitated crystals were filtered and dried, tothereby yield compound 6 (128 mg, 0.341 mmol, 81.6%).

¹H-NMR(CDCl₃) δ7.54(1H, s, H-8), 6.31(1H, t, H-1′, J=7.00), 6.06(1H, dd,H-3′, J=5.00, 6.50), 5.31(2H, bs, NH₂), 4.95(1H, d, H-5′a, J=11.5),4.80(2H, bs, NH₂), 4.37(1H, d, H-5′b, J=12.0), 3.43(1H, m, H-2′a),2.63(1H, m, H-2′b), 2.23, 2.12(each 3H, s, acetyl).

(2) Synthesis of 3′,5′-di-O-acetyl-4′-C-cyano-2′-deoxy-2-fluoroadenosine (Compound 7)

Compound 6 (118 mg, 0.314 mmol) was dissolved in 70% hydrogenfluoride-pyridine (2.30 ml), and t-butyl nitrite (50.0 μl, 0.427 mmol)was added to the resultant solution, followed by stirring at −10° C. forthree hours. To the resultant mixture, t-butyl nitrite (10.0 μl, 85μmol) was further added, and then the mixture was further stirred at−10° C. for one hour. After a saturated aqueous solution of sodiumbicarbonate was added to the resultant mixture, the resultant mixturewas subjected to extraction with ethyl acetate, and the resultantorganic layer was washed with a saturated aqueous solution of sodiumchloride. The resultant organic layer was dried over magnesium sulfate,and then concentrated under reduced pressure. The resultant residue wasdissolved in ethanol under heating, followed by cooling. Thethus-precipitated crystals were filtered and dried, to thereby yieldcompound 7 (53.7 mg, 0.14 mmol, 45.2%).

¹H-NMR(DMSO-d₆)δ8.35(1H, s, H-8), 8.00, 7.92(each 1H, bs, NH₂), 6.54(1H,t, H-1′, J=7.00), 5.83(1H, dd, H-3′, J=4.00, 6.50), 4.63(1H, d, H-5′a,J=11.5), 4.44(1H, d, H-5′b, J=12.0), 3.26(1H, m, H-2′a), 2.73(1H, m,H-2′b), 2.18, 2.05(each 3H, s, acetyl).

(3) Synthesis of 4′-C-cyano-2′-deoxy-2-fluoroadenosine (Compound 8)

Compound 7 (53.7 mg, 0.142 mmol) was dissolved in methanol (1.90 ml),and 28% aqueous ammonia (1.30 ml) was added to the resultant solution,followed by stirring at room temperature for 30 minutes. The resultantreaction mixture was concentrated under reduced pressure, and then theresultant residue was purified by means of silica gel columnchromatography (silica gel 10 ml, hexane/ethyl acetate (5:1), ethylacetate, ethyl acetate/methanol (10:1)), to thereby yield compound 8(30.2 mg, 0.10 mmol, 72.3%).

¹H-NMR(DMSO-d₆) δ8.31(1H, s, H-8), 7.93, 7.82(each 1H, bs, NH₂),6.43(1H, t, H-1′, J=7.00), 6.36(1H, bs, 3′—OH), 5.74(1H, bs, 5′—OH),4.70(1H, t, H-3′, J=5.50), 3.80(1H, d, H-5′a, J=12.0), 3.65(1H, d,H-5′b, J=12.0), 2.93(1H, m, H-2′a), 2.47(1H, m, H-2′b).

Synthesis Example 3 Synthesis of 2-chloro-2′-deoxy-4′-C-ethynyladenosine(Compound 9)

Benzyltriethylammonium nitrite (1.04 g, 4.36 mmol) was dissolved indichloromethane (24.0 ml), and acetyl chloride (0.40 ml, 5.63 mmol) wasadded to the resultant solution, followed by stirring at 0° C. for 30minutes. To the resultant solution, a solution of compound 2 (340 mg,0.91 mmol) in dichloromethane (6.00 ml) was added, and the resultantmixture was stirred at 0° C. for three hours. The resultant reactionmixture was diluted with chloroform, and subsequently the resultantorganic layer was washed with water, dried over anhydrous magnesiumsulfate, and concentrated under reduced pressure. To the resultantresidue, 28% aqueous ammonia (10.0 ml) and methanol (15.0 ml) wereadded, and the resultant mixture was stirred at room temperatureovernight. Thereafter, the resultant reaction mixture was concentratedunder reduced pressure, and the resultant residue was purified by meansof silica gel column chromatography (silica gel 50 ml,chloroform:methanol=20:1 to 10:1). The thus-purified residue was furtherpurified by means of ODS column chromatography (ODS 50 ml, 5 to 10 to 15to 20% acetonitrile), to thereby yield compound 9 (39.2 mg, 0.13 mmol,14.3%).

¹H-NMR(DMSO-d₆) δ8.34(1H, s, H-8), 7.84(2H, bs, NH₂), 6.27(1H, dd, H-1′,J=5.00, 7.00), 5.60(1H, d, 3′—OH, J=5.00), 5.33(1H, t, 5′—OH, J=6.00),4.56(1H, m, H-3′), 3.64(1H, m, H-5′a), 3.56(1H, m, H-5′b), 3.52(1H, s,ethynyl), 2.68(1H, m, H-2′a), 2.45(1H, m, H-2′b).

Synthesis Example 4 Synthesis of 2′-deoxy-4′-C-ethynyl-2-fluoroadenosine5′-H-phosphonate (Compound 10)

Compound 4 (50.0 mg, 0.171 mmol) was dissolved in pyridine (2.00 ml),and phosphonic acid (21.0 mg, 0.25 mmol) and dicyclohexyl carbodiimide(106 mg, 0.51 mmol) were added to the resultant solution, followed bystirring at room temperature for five hours. The resultant precipitatewas removed through filtration, and then the filtrate was concentratedunder reduced pressure. The resultant residue was partitioned with waterand chloroform. The resultant aqueous layer was concentrated underreduced pressure, and the thus-obtained residue was purified by means ofpreparative thin-layer chromatography (isopropanol: 28% aqueousammonia:water=7:1:2). The resultant residue was co-boiled withacetonitrile, and then treated with methanol and ether, to thereby yielda powdery compound (compound 10; 6.3 mg, 17.6 μmmol, 10.3%).

¹H-NMR(D₂O) δ8.13(1H, s, H-8), 6.49(1H, d, H-P, J=645), 6.25(1H, dd,H-1′, J=5.00, 7.50), 3.96(2H, m, H-5′), 2.75, 2.59(each 1H, m, H-2′).³¹P-NMR(D₂O)δ6.45.

Synthesis Example 5 Synthesis of2′,3′-didehydro-2′,3′-dideoxy-4′-C-ethynyl-2-fluoroadenosine (Compound13)

Compound 4 (0.28 g, 0.95 mmol) was dissolved in dimethylformamide (7.00ml), and t-butylchlorodiphenylsilane (0.50 ml, 1.92 mmol) and imidazole(0.26 g, 3.82 mmol) were added to the resultant solution, followed bystirring at room temperature overnight. After methanol was added to theresultant reaction mixture, the resultant mixture was concentrated underreduced pressure, and the resultant residue was partitioned with ethylacetate and water. The resultant organic layer was dried over anhydrousmagnesium sulfate, and then concentrated under reduced pressure. Thethus-obtained residue was purified by means of silica gel columnchromatography (silica gel 100 ml, chloroform methanol=20:1), to therebyyield crude compound 11 (0.38 g).

The crude compound 11 (0.38 g) was dissolved in dichloromethane (10.0ml), and trifluoromethanesulfonic anhydride (0.14 ml, 0.83 mmol) andpyridine (0.14 g, 1.71 mmol) were added to the resultant solution at−10° C., followed by stirring at the same temperature for two hours. Asaturated aqueous solution of sodium bicarbonate was added to theresultant reaction mixture, and then the resultant mixture was subjectedto extraction with chloroform. The resultant organic layer was driedover anhydrous magnesium sulfate, and then concentrated under reducedpressure. The thus-obtained crude triflate was employed in the nextreaction without purification thereof.

The crude triflate was dissolved in dry tetrahydrofuran (20.0 ml), and asolution of 1-M sodium hexamethyldisilazide in tetrahydrofuran (2.50 ml,2.50 mmol) was added to the resultant solution in an argon atmosphere at−78° C., followed by stirring at the same temperature for two hours.Thereafter, the resultant reaction mixture was allowed to warm to roomtemperature, and then stirred overnight. Water was added to theresultant reaction mixture, and then the resultant mixture was subjectedto extraction with ethyl acetate. The resultant organic layer was driedover anhydrous magnesium sulfate, and then concentrated under reducedpressure. The thus-obtained residue was purified by means of silica gelcolumn chromatography (silica gel 50 ml, chloroform : methanol=50:1 to20:1), to thereby yield crude compound 12 (0.20 g).

The thus-obtained crude compound 12 was dissolved in tetrahydrofuran(10.0 ml), and a solution of 1-M tetrabutylammonium fluoride intetrahydrofuran (0.59 ml, 0.59 mmol) was added to the resultantsolution, followed by stirring at room temperature for 30 minutes. Theresultant reaction mixture was concentrated under reduced pressure, andthen a mixture of chloroform and methanol (10:1) was added to thethus-concentrated mixture. The thus-precipitated crystals were filtered,to thereby yield compound 13 (52.0 mg, 0.19 mmol, 20.0% from compound4).

¹H-NMR(DMSO-d₆) δ8.08(1H, s, H-8), 7.84(2H, bs, NH₂), 6.90(1H, t, H-1′,J=1.50), 6.43(1H, dd, H-3′, J=2.00, 6.00), 6.27(1H, dd, H-3′, J=1.00,6.00), 5.37(1H, t, 5′—OH, J=6.00), 3.71(1H, s, ethynyl), 3.67(1H, dd,H-5′a, J=6.00, 12.0), 3.57(1H, dd, H-5′b, J=6.00, 12.0).

Synthesis Example 6 Synthesis of2′,3′-didehydro-2′,3′-dideoxy-4′-C-ethynyl-2-fluoroadenosine5′-H-phosphonate (Compound 14)

Compound 13 (13.0 mg, 0.047 mmol) was dissolved in pyridine (0.7 ml),and phosphonic acid (7.7 mg, 0.094 mmol) and dicyclohexyl carbodiimide(29.2 mg, 0.14 mmol) were added to the resultant solution, followed bystirring at room temperature for one hour. The resultant reactionmixture was concentrated under reduced pressure, and the thus-obtainedresidue was purified by means of ODS column chromatography (ODS 10 ml, 0to 1% acetonitrile). The resultant residue was applied to a Dowex 50W×8columm (Na type) and eluted. The eluate was concentrated, and theresultant residue was treated with methanol and ether, to thereby yielda powdery compound (compound 14; 4.3 mg, 12 μmol, 25.5%).

¹H-NMR(MeOD) δ8.30(1H, s, H-8), 6.69(1H, d, H-P, J=625), 7.02(1H, bt,H-1′), 6.48(1H, dd, H-2′, J=2.00, 5.50), 6.22(1H, dd, H-3′, J=1.00,5.50), 4.18(1H, dd, H-5′a, J=7.50, 11.0), 3.99(1H, dd, H-5′b, J=7.50,11.0). ³¹P-NMR(MeOD) δ4.11.

Synthesis Example 7 Synthesis of2′,3′-dideoxy-4′-C-ethynyl-2-fluoroadenosine (Compound 23)

(1) Synthesis of1,2-O-isopropylidene-4-C-triethylsilylethynyl-α-D-xylo-pentofuranose(Compound 16)

Oxalyl chloride (0.54 ml, 6.19 mmol) was dissolved in dichloromethane(10.0 ml), and then dimethyl sulfoxide (0.90 ml, 12.7 mmol) was addeddropwise to the resultant solution at −60° C., followed by stirring atthe same temperature for 15 minutes. A solution of compound 15 (1.06 g,3.13 mmol, Biosci. Biotech. Biochem., 57, 1433-1438 (1993)) indichloromethane (15.0 ml) was added dropwise to the resultant mixture,followed by stirring at −60° C. for 30 minutes. After triethylamine(1.86 ml, 13.3 mmol) was added thereto, the resultant reaction mixturewas allowed to warm to room temperature, followed by stirring for 30minutes. The reaction mixture was diluted with chloroform, and thenwashed with water. The thus-obtained organic layer was dried overanhydrous magnesium sulfate, and then concentrated under reducedpressure. The thus-obtained crude aldehyde was employed in the nextreaction without purification thereof.

The crude aldehyde was dissolved in dichloromethane (40.0 ml), andcarbon tetrabromide (2.08 g, 6.27 mmol) and triphenylphosphine (3.28 g,12.5 mmol) were added to the resultant solution at 0° C., followed bystirring at room temperature for one hour. After triethylamine (2.60 ml,18.7 mmol) was added to the resultant reaction mixture, the resultantmixture was diluted with chloroform, and the resultant organic layer waswashed with water. The organic layer was dried over anhydrous magnesiumsulfate, and then concentrated under reduced pressure. The resultantresidue was purified by means of silica gel column chromatography(silica gel 100 ml, hexane:ethyl acetate=3:1), to thereby yield a crudedibromoethene (1.42 g).

The crude dibromoethene (1.42 g, 2.89 mmol) was dissolved in drytetrahydrofuran (20.0 ml), and a solution of 2.2-M methyllithium inether (4.49 ml, 9.88 mmol) was added to the resultant solution in anargon atmosphere at −10° C., followed by stirring at the sametemperature for five minutes. Chlorotriethylsilane (0.95 ml, 5.66 mmol)was added to the resultant mixture, and the mixture was further stirredfor 30 minutes. After a saturated aqueous solution of ammonium chloridewas added to the resultant reaction mixture, the resultant mixture wasstirred, and then subjected to extraction with ethyl acetate. Theresultant organic layer was dried over anhydrous magnesium sulfate, andthen concentrated under reduced pressure, to thereby yield a crudealkyne.

The crude alkyne was dissolved in acetic acid (80.0 ml), and water (20.0ml) was added to the resultant solution, followed by stirring at roomtemperature overnight. The resultant reaction mixture was concentratedunder reduced pressure, and the resultant residue was co-boiled withtoluene. The resultant residue was purified by means of silica gelcolumn chromatography (silica gel 50 ml, hexane ethyl acetate=3:1), tothereby yield compound 16 (0.70 g, 2.13 mmol, 64.4%).

¹H-NMR(CDCl₃) δ6.00(1H, d, H-1, J=3.50), 4.60(1H, d, H-2, J=4.00),4.58(1H, d, H-3, J=5.00), 3.96-3.91(3H, m, H-5 and 3—OH), 2.50(1H, t,5—OH), 1.64, 1.33(each 3H, s, acetonide), 0.97(9H, t, Et, J=8.00),0.59(6H, q, Et, J=8.00).

(2) Synthesis of5-O-t-butyldiphenylsilyl-1,2-O-isopropylidene-4-C-triethylsilylethynyl-α-D-xylo-pentofuranose(Compound 17)

Compound 16 (0.70 g, 2.13 mmol) was dissolved in dimethylformamide (3.50ml), and t-butylchlorodiphenylsilane (0.66 ml, 2.54 mmol) and imidazole(0.35 g, 5.14 mmol) were added to the resultant solution, followed bystirring overnight. After methanol was added to the resultant reactionmixture, and the resultant mixture was concentrated under reducedpressure, the thus-obtained residue was dissolved in ethyl acetate. Theresultant organic layer was washed with water, and then dried overanhydrous magnesium sulfate, and concentrated under reduced pressure.The resultant residue was purified by means of silica gel columnchromatography (silica gel 100 ml, hexane:ethyl acetate=5:1), to therebyyield compound 17 (1.10 g, 1.94 mmol, 91.1%).

¹H-NMR(CDCl₃) δ7.72-7.36(10H, s, aromatic), 6.02(1H, d, H-1, J=3.50),4.66(1H, d, H-3, J=5.50), 4.63(1H, d, H-1, J=4.00), 4.05(1H, d, H-5a,J=10.5), 3.99(1H, d, 3—OH, J=5.50), 3.93(1H, d, H-5′b, J=10.5), 1.65,1.35(each 3H, s, acetonide), 1.06(9H, s, t-Bu), 0.93(9H, t, Et, J=8.00),0.56(6H, q, Et, J=8.00).

(3) Synthesis of5-O-t-butyldiphenylsilyl-3-deoxy-1,2-O-isopropylidene-4-C-triethylsilylethynyl-α-D-xylo-pentofuranose(Compound 18)

Compound 17 (1.10 g, 1.94 mmol) was dissolved in acetonitrile (20.0 ml),and phenyl chlorothionoformate (0.40 ml, 2.89 mmol) and4-dimethylaminopyridine (0.71 g, 5.81 mmol) were added to the resultantsolution, followed by stirring at room temperature for three hours. Theresultant reaction mixture was diluted with ethyl acetate, and then theresultant organic layer was washed with 0.1-N hydrochloric acid and asaturated aqueous solution of sodium bicarbonate. The resultant organiclayer was dried over anhydrous magnesium sulfate, and concentrated underreduced pressure. The thus-obtained crude thiocarbonate was employed inthe next reaction without purification thereof.

The crude thiocarbonate was co-boiled with toluene three times, and thendissolved in toluene (30.0 ml), followed by degassing under reducedpressure. Tributyltin hydride (2.61 ml, 9.70 mmol) and a small amount ofazobis(isobutyronitrile) were added to the resultant solution in anargon atmosphere at 80° C., and the resultant mixture was stirred underthe same conditions for one hour. The resultant reaction mixture wasconcentrated under reduced pressure, and then the thus-obtained residuewas purified by means of silica gel column chromatography (silica gel100 ml, hexane:ethyl acetate=10:1), to thereby yield compound 18 (1.07g, 1.94 mmol, quant.).

¹H-NMR(CDCl₃) δ7.69-7.38(10H, m, aromatic), 5.90(1H, d, H-1, J=4.00),4.85(1H, t, H-2, J=5.00), 3.82(1H, d, H-5a, J=11.0), 3.58(1H, d, H-5b,J=10.5), 2.64(1H, dd, H-3a, J=6.00, 14.0), 2.40(1H, d, H-3b, J=14.0),1.68, 1.36(each 3H, s, acetonide), 1.04(9H, s, t-Bu)0.92(9H, t, Et,J=8.00), 0.54(6H, q, Et, J=8.00).

(4) Synthesis of1,2-di-O-acetyl-5-O-t-butyldiphenylsilyl-3-deoxy-4-C-triethylsilylethynyl-D-xylo-pentofuranose(Compound 19)

Compound 18 (1.07 g, 1.94 mmol) was dissolved in 80% acetic acid (100ml), and trifluoroacetic acid (10.0 ml) was added to the resultantsolution, followed by stirring at 40° C. for three hours. The resultantreaction mixture was concentrated under reduced pressure, and then thethus-obtained residue was co-boiled with toluene. The resultant residuewas purified by means of silica gel column chromatography (silica gel100 ml, hexane:ethyl acetate=4:1). The resultant residue was dissolvedin pyridine (20.0 ml), and acetic anhydride (0.49 ml) was added to theresultant solution, followed by stirring at room temperature overnight.The resultant reaction mixture was concentrated under reduced pressure,and then the thus-obtained residue was co-boiled with toluene. Theresultant residue was purified by means of silica gel columnchromatography (silica gel 100 ml, hexane:ethyl acetate=5:1), to therebyyield compound 19 (0.75 g, 1.26 mmol, 64.9%).

¹H-NMR(CDCl₃) δ7.71-7.37(10H, m, aromatic), 6.44(0.3H, d, H-1-alpha,J=4.50), 6.30(0.7H, s, H-1-beta), 5.36(0.3H, m, H-2-alpha), 5.19(0.7H,d, H-2-beta, J=5.50), 3.77, 3.74(each 0.7H, d, H-5-beta, J=10.0), 3.76,3.62(each 0.3H, d, H-5-alpha, J=11.0), 2.86(0.3H, dd, H-3a-alpha,J=8.50, 12.5), 2.72(0.7H, dd, H-3a-beta, J=5.50, 14.0), 2.39(0.3H, dd,H-3b-alpha, J=10.0, 12.5), 2.33(0.7H, d, H-3b-beta, J=14.0), 2.11,2.08(each 0.9H, s, acetyl-alpha), 2.10, 1.80(each 2.1H, s, acetyl-beta),1.074(6.3H, s, t-Bu-beta), 1.067(2.7H, s, t-Bu-alpha), 0.97(6.3H, t,Et-beta, J=8.00), 0.94(2.7H, t, Et-alpha, J=8.00), 0.58(4.2H, q,Et-beta, J=8.00), 0.54(1.8H, q, Et-alpha, J=8.00).

(5) Synthesis of9-(2-O-acetyl-5-O-t-butyldiphenylsilyl-3-deoxy-4-C-triethylsilylethynyl-β-D-xylo-pentofuranosyl)-2,6-diaminopurine(Compound 20)

2,6-Diaminopurine (1.21 g, 8.06 mmol) was suspended in acetonitrile(24.0 ml), and N,O-bis(trimethylsilyl)acetamide (11.9 ml, 48.1 mmol) wasadded to the resultant suspension, followed by stirring at 80° C. forthree hours. The resultant solution was concentrated under reducedpressure, and then the thus-obtained residue was co-boiled with1,2-dichloroethane three times. To the resultant residue, a solution ofcompound 19 (2.39 g, 4.02 mmol) in 1,2-dichloroethane (24.0 ml), andtrimethylsilyl trifluoromethanesulfonate (3.05 ml, 16.9 mmol) wereadded, and the resultant mixture was stirred in an argon atmosphere at50° C. for five hours and at 80° C. for 10 hours. After a saturatedaqueous solution of sodium bicarbonate was added to the resultantmixture, and then the mixture was stirred, the resultant solution wasfiltered through celite. The resultant organic layer was dried overanhydrous magnesium sulfate, and then concentrated under reducedpressure. The thus-obtained residue was purified by means of silica gelcolumn chromatography (silica gel 300 ml, chloroform methanol=20:1). Thethus-purified residue was crystallized from hexane and ethyl acetate, tothereby yield compound 20 (1.60 g, 2.34 mmol, 58.2%).

¹H-NMR(CDCl₃) δ7.67-7.33(10H, m, aromatic), 6.13(1H, d, H-1′, J=3.50),5.77(1H, md, H-2′), 5.28(2H, bs, NH₂), 4.49(2H, bs, NH₂), 3.67(1H, d,H-5′a, J=10.5), 3.79(1H, d, H-5′b, J=11.0), 3.18(1H, dd, H-3′a, J=7.50,14.0), 2.37(1H, dd, H-3′b, J=3.00, 14.0), 1.06(9H, s, t-Bu), 0.99(9H, t,Et, J=8.00), 0.61(6H, q, Et, J=8.00).

(6) Synthesis of5′-O-t-butyldimethylsilyl-3′-deoxy-4′-C-triethylsilylethynyl-2-fluoroadenosine(Compound 21)

Compound 20 (100 mg, 0.146 mmol) was dissolved in pyridine (3.00 ml),and hydrogen fluoride-pyridine (7.00 ml) and t-butyl nitrite (160 μl,1.34 mmol) were added to the resultant solution at −15° C., followed bystirring at the same temperature for 30 minutes. After water was addedto the resultant reaction mixture, the resultant mixture was subjectedto extraction with ethyl acetate. The resultant organic layer was washedwith a saturated aqueous solution of sodium bicarbonate, and then driedover anhydrous magnesium sulfate and concentrated under reducedpressure. The thus-obtained residue was purified by means of silica gelcolumn chromatography (silica gel 300 ml, chloroform:methanol=100:1 to20:1). The resultant residue (48.9 mg) was dissolved in dichloromethane(1.30 ml), and t-butyldimethylsilyl trifluoromethanesulfonate (36.0 μl,0.157 mmol) and collidine (44.1 μl, 0.0331 mmol) were added to theresultant solution at 0° C., followed by stirring at the sametemperature for 50 minutes. Water was added to the resultant reactionmixture, and the resultant mixture was subjected to extraction withchloroform. The resultant organic layer was washed with 0.01-Nhydrochloric acid and a saturated aqueous solution of sodiumbicarbonate, and then dried over anhydrous magnesium sulfate andconcentrated under reduced pressure. The resultant residue was dissolvedin dioxane (3.00 ml), and 28% aqueous ammonia (0.30 ml) was added to theresultant solution, followed by stirring at room temperature for 30minutes. The resultant reaction mixture was concentrated under reducedpressure, and then the thus-obtained residue was dissolved in methanol(1.20 ml), and 28% aqueous ammonia (0.80 ml) was added to the resultantsolution, followed by stirring at room temperature for two hours. Theresultant reaction mixture was concentrated under reduced pressure, andthe thus-precipitated crystals were recovered through filtration, tothereby yield compound 21 (34.0 mg, 0.0652 mmol, 44.7%).

¹H-NMR(CDCl₃) δ8.04(1H, s, H-8), 5.99(1H, d, H-1′, J=4.00), 5.80(2H, bs,NH₂), 4.73(1H, m, H-2′), 4.23(1H, d, 2′—OH, J=5.00), 3.88(1H, d, H-5′a,J=11.0), 3.70(1H, d, H-5′b, J=11.0), 2.82(1H, dd, H-3′a, J=7.50, 13.0),2.42(1H, dd, H-3′b, J=6.50, 13.0), 1.01(9H, t, Et, J=8.00), 0.80(9H, s,t-Bu), 0.64(6H, q, Et, J=8.00), 0.039, −0.013(each 3H, s, Me).

(7) Synthesis of5′-O-t-butyldimethylsilyl-2′,3′-dideoxy-4′-C-triethylsilylethynyl-2-fluoroadenosine(Compound 22)

Compound 21 (32.0 mg, 0.061 mmol) was co-boiled with acetonitrile threetimes, and then dissolved in acetonitrile (1.00 ml). To the resultantsolution, phenyl chlorothionoformate (12.7 μl, 0.092 mmol) and4-dimethylaminopyridine (22.5 mg, 0.180 mmol) were added, and theresultant mixture was stirred at room temperature for one hour. Theresultant reaction mixture was diluted with ethyl acetate, and then thethus-obtained organic layer was washed with 0.01-N hydrochloric acid anda saturated aqueous solution of sodium bicarbonate, and dried overanhydrous magnesium sulfate. The resultant organic layer wasconcentrated under reduced pressure, and the thus-obtained crudethiocarbonate was employed in the next reaction without purificationthereof.

The crude thiocarbonate was co-boiled with toluene three times, and thendissolved in toluene (1.00 ml), followed by degassing under reducedpressure. To the resultant solution, tris(trimethylsilyl)silane (94.6μl, 0.306 mmol) and a small amount of azobis(isobutyronitrile) wereadded in an argon atmosphere at 80° C., and the resultant mixture wasstirred under the same conditions for one hour. The resultant reactionmixture was concentrated under reduced pressure, and then thethus-obtained residue was purified by means of silica gel columnchromatography (silica gel 10 ml, chloroform:methanol=200:1 to 100:1),to thereby yield compound 22 (26.1 mg, 0.0516 mmol, 84.6%).

¹H-NMR(CDCl₃) δ8.24(1H, s, H-8), 6.36(1H, dd, H-1′, J=2.50, 7.00),5.91(2H, bs, NH₂), 4.04(1H, d, H-5′a, J=11.0), 3.81(1H, d, H-5′b,J=11.0), 2.83(1H, m, H-2′a), 2.54(1H, m, H-3′a), 2.37(1H, m, H-2′b),2.11(1H, m, H-3′b), 1.00(9H, t, Et, J=8.00), 0.93(9H, s, t-Bu), 0.62(6H,q, Et, J=8.00), 0.13(6H, s, Me).

(8) Synthesis of 2′,3′-dideoxy-4′-C-ethynyl-2-fluoroadenosine (Compound23)

Compound 22 (101 mg, 0.200 mmol) was dissolved in tetrahydrofuran (10ml), and a solution of 1-M tetrabutylammonium fluoride intetrahydrofuran (0.42 ml, 0.42 mmol) was added to the resultantsolution, followed by stirring at room temperature for five minutes.After acetic acid (24 μl) was added to the resultant reaction mixture,the resultant mixture was concentrated under reduced pressure. Thethus-obtained residue was purified by means of silica gel columnchromatography (silica gel 15 ml, chloroform methanol=40:1 to 20:1), tothereby yield compound 23 (53.0 mg, 0.191 mmol, 95.7%).

¹H-NMR(MeOD) δ8.23(1H, s, H-8), 6.22(1H, dd, H-1′, J=4.00, 7.00),3.77(1H, d, H-5′a, J=12.5), 3.61(1H, d, H-5′b, J=12.0), 2.94(1H, s,ethynyl), 2.66(1H, m, H-2′a), 2.54(1H, m, H-3′a), 2.42(1H, m, H-2′b),2.11(1H, m, H-3′b).

Synthesis Example 8 Synthesis of2′,3′-dideoxy-4′-C-ethynyl-2-fluoroadenosine 5′-H-phosphonate (Compound24)

Compound 23 (20.0 mg, 0.07 mmol) was dissolved in pyridine (1 ml), andphosphonic acid (11.8 mg, 0.144 mmol) and dicyclohexyl carbodiimide(44.7 mg, 0.216 mmol) were added to the resultant solution, followed bystirring at room temperature for five hours. The resultant reactionmixture was concentrated under reduced pressure, and the thus-obtainedresidue was purified by means of ODS column chromatography (ODS 10 ml, 0to 1% acetonitrile). The resultant residue was applied to a Dowex 50W×8columm (Na type) and eluted. The eluate was concentrated, and theresultant residue was treated with methanol and ether, to thereby yielda powdery compound (compound 24; 4.7 mg, 13 μmol, 18.6%).

¹H-NMR(MeOD) δ8.37(1H, s, H-8), 6.77(1H, d, H-P, J=625), 6.32(1H, dd,H-1′, J=4.00, 6.50), 4.09(1H, m, H-5′), 2.71(2H, m, H-2′a, H-3′a),2.52(1H, m, H-2′b), 2.29(1H, m, H-3′b). ³¹P-NMR(MeOD) δ4.52.

Drug Preparation Example 1 Tablets

Compound of the present invention 30.0 mg Cellulose micropowder 25.0 mgLactose 39.5 mg Starch 40.0 mg Talc  5.0 mg Magnesium stearate  0.5 mg

Tablets are prepared from the above composition through a customarymethod.

Drug Preparation Example 2 Capsules

Compound of the present invention 30.0 mg Lactose 40.0 mg Starch 15.0 mgTalc  5.0 mg

Capsular drugs are prepared from the above composition through acustomary method.

Drug Preparation Example 3 Injections

Compound of the present invention  30.0 mg Glucose 100.0 mg

Injections are prepared by dissolving the above composition in purifiedwater for preparing injections.

Test Examples will next be described. Employed in tests were thefollowing five compounds of the present invention and four knowncompounds.

Invention Compounds:

Compound 4: 2′-deoxy-4′-C-ethynyl-2-fluoroadenosine;

Compound 8: 4′-C-cyano-2′-deoxy-2-fluoroadenosine;

Compound 9: 2-chloro-2′-deoxy-4′-C-ethynyladenosine;

Compound 10: 2′-deoxy-4′-C-ethynyl-2-fluoroadenosine 5′-H-phosphonate;and

Compound 13:2′,3′-didehydro-2′,3′-dideoxy-4′-C-ethynyl-2-fluoroadenosine.

Known Compounds:

AZT: Azidothymidine;

EdAdo: 2′-deoxy-4′-C-ethynyladenosine;

EdDAP: 9-(4-C-ethynyl-2-deoxy-ribopentofuranosyl)-2,6-diaminopurine; and

ddAdo: 2′,3′-dideoxyadenosine.

Test Example 1

<Test methods> Anti-Human-Immunodeficiency-Virus (HIV) Activity

1) MTT Method Using MT-4 Cells

-   1. A test agent (100 μl) is diluted on a 96-well microplate. MT-4    cells infected with HIV-1 (IIIb strain; 100 TCID₅₀) and non-infected    MT-4 cells are added to the microplate such that the number of cells    in each well becomes 10,000. The cells are cultured at 37° C. for    five days.-   2. MTT (20 μl, 7.5 mg/ml) is added to each well, and the cells are    further cultured for 2-3 hours.-   3. The cultured medium (120 μl) is sampled, and MTT terminating    solution (isopropanol containing 4% Triton X-100 and 0.04N HCl) is    added to the sample. The mixture is stirred to dissolve formed    formazan. The absorbance at 540 nm of the solution is measured.    Since the absorbance is proportional to the number of viable cells,    the test agent concentration at which a half value of the absorbance    is measured in a test using infected MT-4 cells represents EC₅₀,    whereas the test agent concentration at which a half value of the    absorbance is measured in a test using non-infected MT-4 cells    represents CC₅₀.    2) MAGI Assay Using HeLa CD4/LTR-Beta-Ga1 Cells-   1. HeLa CD4/LTR-beta-Ga1 cells are added to 96 wells such that the    number of cells in each well is 10,000. After 12-24 hours, the    culture medium is removed, and a diluted test agent (100 μl) is    added.-   2. A variety of HIV strains (wild strain: WT, drug-resistant strain:    MDR and M184V; each being equivalent to 50 TCID₅₀) are added, and    the cells are further cultured for 48 hours.-   3. The cells are fixed for five minutes using PBS supplemented with    1% formaldehyde and 0.2% glutaraldehyde.-   4. After the fixed cells are washed with PBS three times, the cells    are stained with 0.4 mg/ml X-Ga1 for one hour, and the number of    blue-stained cells of each well is counted under a transmission    stereoscopic microscope. The test agent concentration at which    blue-stained cells decrease to 50% in number represents EC₅₀.    <Results> Anti-Human-Immunodeficiency Virus (HIV) Activity and    Cytotoxicity    1) MTT Method Using MT-4 Cells

TABLE 1 MT-4 cells Anti-HIV-1 activity Cytotoxicity Selectivity IndexDrugs (EC₅₀, μM) (CC₅₀, μM) (CC₅₀/EC₅₀) Compound 4 0.000068 7.5 110000EdDAP 0.00034 0.9 2600 EdAdo 0.0098 16 1630 AZT 0.0032 29.4 91902) MAGI Assay Using HeLa CD4/LTR-Beta-Ga1 Cells

TABLE 2 HeLa CD4/LTR-beta-Gal cells Anti-HIV-1_(wild) Anti-HIV-1_(MDR)Anti-HIV-1_(M184V) activity activity activity Drugs (EC₅₀, μM) (EC₅₀,μM) (EC₅₀, μM) Compound 4 0.00020 0.0001448 0.003107 Compound 8 0.120.95 4.8 Compound 9 0.0019 0.0084 0.01 Compound 10 0.0034 0.003 0.062Compound 13 0.80 0.15 1.8 EdAdo 0.008 0.0062 0.047 AZT 0.022 15.3 0.01

Test Example 2

<Test Methods> Stability of Compound 4 Against Adenosine Deaminase

Calf-intestine-derived adenosine deaminase (0.01 unit) was added to 0.5ml of 0.5-mM compound 4 (50 mM Tris-HCl buffered solution (pH 7.5)), andthe mixture was incubated at 25° C.

A 5-μl aliquot of the reaction mixture was removed every 15 minutes,followed by analysis by means of HPLC (high performance liquidchromatography). The peak area of a test drug at reaction time 0 wastaken as 100%, and the curve was monitored over time. The HPLC analysiswas performed under the following conditions.

Column: YMC-Pack ODS-A (250×6.0 mm)

Eluent: 15% MeCN-50 mM TEAA

Flow rate: 1 mL/min.

Temperature: 30° C.

Detection: 260 nm

<Results>

As shown in FIG. 1, 2′-deoxy-4′-C-ethynyl-2-fluoroadenosine, which isCompound 4 of the present invention, was not at all deaminated, ascontrasted to the case where conventional 2′-deoxy-4′-C-ethynyladenosine(EdAdo) was deaminated, proving that the compound of the presentinvention has resistance to adenosine deaminase.

Test Example 3

<Test Methods> Stability of Compound 4 under Acidic Conditions

Compound 4 (2.9 mg) or 2′,3′-dideoxyadenosine (ddAdo: 2.4 mg) wasdissolved in 10 ml of a 37° C. test solution (which had been prepared byadding 2.0 g of sodium chloride and 7.0 ml of hydrochloric acid intowater to make a solution of 1,000 ml), followed by incubation at thesame temperature (37° C.)

A 100-μl aliquot of the reaction mixture was removed therefrom, andneutralized with aqueous 0.1-N sodium hydroxide solution, followed byanalysis of 5 μl by means of HPLC. The HPLC analysis conditions are thesame as those employed in Test Example 2.

<Results>

About 98% of ddAdo, which is a conventional compound, is degraded inabout five minutes under the above conditions (see FIG. 3), whereas2′-deoxy-4′-C-ethynyl-2-fluoroadenosine, which is Compound 4 of thepresent invention, was degraded very slowly, proving that the compoundof the present invention is relatively stable under acidic conditions(see FIG. 2).

Test Example 4

<Test Methods> In Vivo Acute Toxicity Test of Compound 4

Groups of ICR mice (6 weeks of age, male), each group consisting of 8mice, were given a test drug (Compound 4; dissolved or suspended insaline) via oral route or intravenous injection in amounts up to 100mg/kg. The occurrence of death and body weight of each mouse weremonitored for seven days.

<Results>

All the mice to which Compound 4 was administered up to 100 mg/kg in asingle dose survived regardless of the administration route of oral orintravenous (Table 3). Also, as shown in FIG. 4, weight loss andpathological symptoms such as diarrhea were not observed. Thus, it hasnow been confirmed that 2′-deoxy-4′-C-ethynyl-2-fluoroadenosine(Compound 4) of the present invention does not exhibit acute toxicity inmice.

TABLE 3 Survisors/Total Dose (mg/kg) Oral Intravenous Placebo 8/8 8/8 18/8 8/8 3 8/8 8/8 10  8/8 8/8 30  8/8 8/8 100  8/8 8/8

1. A 4′-C-substituted-2-haloadenosine derivative, which is2′-deoxy-4′-C-ethynyl-2-fluoroadenosine.
 2. A4′-C-substituted-2-haloadenosine derivative, which is4′-C-cyano-2′-deoxy-2-fluoroadenosine.
 3. A4′-C-substituted-2-haloadenosine derivative, which is2-chloro-2′-deoxy-4′-C-ethynyladenosine.
 4. A4′-C-substituted-2-haloadenosine derivative, which is2′-deoxy-4′-C-ethynyl-2-fluoroadenosine 5′-H-phosphonate.
 5. A4′-C-substituted-2-haloadenosine derivative, which is2′,3′-didehydro-2′,3′-dideoxy-4′-C-ethynyl-2-fluoroadenosine.
 6. A4′-C-substituted-2-haloadenosine derivative, which is 2′,3′-didehydro-2′,3′-dideoxy-4′-C-cyano-2-fluoroadenosine.
 7. A4′-C-substituted-2-haloadenosine derivative, which is2′,3′-didehydro-2′,3′-dideoxy-4′-C-ethynyl-2-chloroadenosine.
 8. A4′-C-substituted-2-haloadenosine derivative, which is2′,3′-dideoxy-4′-C-ethynyl-2-fluoroadenosine.
 9. A4′-C-substituted-2-haloadenosine derivative, which is2′,3′-dideoxy-4′-C-cyano-2-fluoroadenosine.
 10. A4′-C-substituted-2-haloadenosine derivative, which is2′,3′-dideoxy-4′-C-ethynyl-2-chloroadenosine.